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. Author manuscript; available in PMC: 2020 Jan 1.
Published in final edited form as: Mol Cancer Res. 2018 Aug 14;17(1):165–176. doi: 10.1158/1541-7786.MCR-18-0399

Increased genetic instability and accelerated progression of colitis-associated colorectal cancer through intestinal epithelium-specific deletion of Klf4

Vincent W Yang 1,2,*, Yang Liu 1, Julie Kim 1, Kenneth R Shroyer 3, Agnieszka B Bialkowska 1,*
PMCID: PMC6318049  NIHMSID: NIHMS1503910  PMID: 30108164

Abstract

Krüppel-like factor 4 (KLF4), a zinc finger transcription factor, regulates homeostasis of the intestinal epithelium. Previously, it was reported that KLF4 functions as a tumor suppressor in colorectal cancer. Here, evidence demonstrates that KLF4 mitigates the development and progression of colitis-associated colorectal cancer (CAC) in a murine model. Mice with intestinal epithelium-specific deletion of Klf4 (Klf4ΔIS) and control mice (Klf4fl/fl) were used to explore the role of KLF4 in the development of azoxymethane (AOM) and dextran sodium sulfate (DSS)-induced CAC. Upon AOM and DSS treatment, KLF4 expression was progressively lost in colonic tissues of Klf4fl/fl mice during tumor development. Klf4ΔIS mice treated with AOM/DSS developed significantly more adenomatous polyps and carcinomas in situ in comparison to treated Klf4fl/fl mice. Adenomatous polyps, but not normal-appearing mucosa, from colonic tissues of treated Klf4ΔIS mice contained a significantly increased number of mitotic cells with more than 2 centrosomes relative to treated control mice. KLF4 and p53 co-localize to the centrosomes in mouse embryonic fibroblasts (MEFs). Absence of KLF4 in Klf4−/− MEFs inhibits and its overexpression restores p53 localization to the centrosomes in Klf4−/− MEFs.

Keywords: KLF4, p53, colitis-associated carcinogenesis, centrosome hyper-amplification

Introduction

Epidemiologic data indicate that inflammatory bowel disease (IBD) is associated with increased risks of colorectal cancer (1, 2). In IBD, colitis-associated colorectal cancer (CAC) progresses in a stepwise fashion from low-grade dysplasia in the background of chronic inflammation to high-grade dysplasia and eventually to invasive carcinoma, in a sequence referred to as the inflammation–dysplasia–carcinoma pathway (3). As in the case of sporadic colorectal cancer, chromosomal instability, microsatellite instability, and promoter hypermethylation have been implicated in CAC carcinogenesis (4–6). Recent evidence also suggests that inflammation-induced environmental changes in the gut are fundamental in the induction of CAC (7). Despite these advances, the exact mechanisms that modulate development and progression of CAC remain unclear.

Previous studies demonstrated that Krüppel-like factor 4 (KLF4), a member of a conserved family of zinc finger transcription factors, plays an important role in regulating homeostasis of the intestinal epithelium (8–10). Several studies also reported that KLF4 levels are reduced in colorectal cancer tissues comparing to the paired normal mucosa (11–13). In two independent murine models of colorectal cancer, we showed that KLF4 suppressed development and progression of intestinal neoplasia (14). In each model, KLF4 was shown to influence, to some extent, the genetic and epigenetic modifications and the inflammatory response. This was substantiated by the finding that Klf4 deletion in the intestinal epithelium protected mice from inflammation induced by dextran sulfate sodium (DSS) by modulating the nuclear factor kappa B (NF-κB) pathway (15). Finally, our previous studies showed that KLF4 plays a crucial role in the maintenance of genetic stability by regulating chromosome and centrosome duplications (16–18). Here we investigated the role of KLF4 in the carcinogenesis of a mouse model of CAC resulted from AOM and DSS treatment.

Materials and Methods

Mice

All animal studies were approved by the Stony Brook University Institutional Animal Care and Use Committee (IACUC) and performed in accordance with institutional policies and NIH guidelines. Mice with the floxed Klf4 gene (Klf4fl/fl) and intestinal epithelium-specific villin-Cre-driven Klf4 deletion (Klf4ΔIS) were described previously (10).

Azoxymethane and dextran sodium sulfate treatment

Adult gender- and age-matched Klf4fl/fl and Klf4ΔIS mice were injected intraperitoneally (IP) with 10 mg/kg of AOM working solution. After 7 days, normal water was replaced with 2.5% DSS in the drinking water for 5 days, followed by 2 weeks of recovery (with regular water). This was followed by a second cycle of 2.5% DSS for 5 days, with 2 weeks of recovery (with regular water). The mice were euthanized at the end of the last recovery treatment, and samples were collected for pathological analysis. Mice were weighed every other day during the treatment.

Tissue harvesting and tumor assessment

Tissues were collected as described previously (19). The colonic tissues were examined under a dissecting microscope for the presence of adenomas. The number and size of adenomas were recorded as described previously (20).

Tissue preparation and immunostaining

The colonic tissues were prepared for the immunohistochemistry (IHC) and immunostaining protocol as described previously (19). Tissue sections were baked in a 65°C oven overnight and subsequently deparaffinized in xylene, incubated in a 2% hydrogen peroxide in methanol bath, and then rehydrated by incubation in a decreasing ethanol bath series (100%, 95%, and 70%), followed by antigen retrieval in citrate buffer solution (10 mM sodium citrate, 0.05% Tween-20, pH 6.0) at 120°C for 10 minutes using a decloaking chamber (Biocare Medical) and 30 minutes incubation at 4°C. The histological sections were incubated with blocking buffer (5% bovine serum albumin and 0.01% Tween 20 in 1X Tris-buffered phosphate-buffered saline [TTBS]) for 1 hour at 37°C. The primary antibodies goat anti-KLF4 (1:300; R&D: AF3158); rabbit anti-cyclin D1 (1:200; Biocare Medical: CRM307AK); rabbit anti-phospho-β-catenin serine 552 (1:200; Cell Signaling: 9566S); rabbit anti-phospho-p44/42 MAPK (p-ERK1/2) (1:200; Cell Signaling: 9101S); mouse anti-p65 (1:500, Santa Cruz Biotechnology: sc-109); rat anti-MKI67 (1:300; DAKO: M7249); mouse anti-α-tubulin (1:250, Sigma-Aldrich: T9026); and goat anti-γ-tubulin FITC-conjugated (1:100, Santa Cruz Biotechnology: sc-7396 FITC) were added and incubated at 4°C overnight. IHC staining of cyclin D1, phospho-β-catenin serine 552, p-ERK1/2, and p65 was performed using goat anti-mouse or anti-rabbit HRP-labeled (Jackson ImmunoResearch Laboratories: 115–035-174 and 111–035-144, respectively) secondary antibody at a 1:500 dilution in blocking buffer for 30 minutes at 37°C, followed by washing and Betazoid DAB (Biocare Medical: BDB2004L) development. For KLF4, secondary unconjugated rabbit anti-goat antibody and goat anti-rabbit antibody (Jackson ImmunoResearch Laboratories: 305–005-003 and 111–005-144, respectively) were added at 1:500 dilution in blocking buffer for 30 minutes at 37°C. After washing, donkey anti-goat HRP-labeled antibodies (Jackson ImmunoResearch Laboratories: 705–035-147) were then added at 1:500 dilution in blocking buffer for 30 minutes at 37°C, followed by Betazoid DAB (Biocare Medical: BDB2004L) development. Detection of all other primary antibodies for IHC was carried out using either Mach3 rabbit or Mach3 mouse HRP-polymer detection (Biocare Medical: M3R531H and M3M530G, respectively). All IHC staining was followed by hematoxylin counterstaining, incubation with ethanol bath (70%, 95%, 100%), xylene, and mounting with Cytoseal XYL xylene-based mounting media (Thermo Scientific: 8312–4). For IF staining, appropriate Alexa Fluor–labeled secondary antibodies (Molecular Probes) were added at 1:500 dilution in blocking buffer for 30 minutes at 37°C, counterstained with Hoechst 33258 (ThermoFisher Scientific: H3569), mounted with Fluoromount Aqueous Mounting Medium (Sigma-Aldrich: F4680), and coverslipped. Slides were analyzed using a Nikon eclipse 90i microscope (Nikon Instruments Inc.) equipped with DS-Qi1Mc and DS-Fi1 CCD cameras (Nikon Instruments Inc.).

H&E staining

Histology of sections was observed on stained 5-μm sections that were fixed, paraffin embedded, deparaffinized, and rehydrated as mentioned previously. Then, they were stained with Hematoxylin Stain Solution, Gill 3 (Ricca Chemical Company: 3537–32) and Eosin Y (Sigma-Aldrich: HT110216). Sections were dehydrated in an increasing series of ethanol baths (70%, 95%, and 100%), cleared in xylene, and mounted with Cytoseal XYL xylene-based mounting media (Thermo Scientific: 8312–4). The H&E stains were used for histopathological assessment.

MKI67+ cells analysis in vivo

Colonic sections of adenomatous polyps and carcinomas in situ of Klf4fl/fl and Klf4ΔIS mice after AOM/DSS treatment were stained with Hoechst (ThermoFisher Scientific: H3569) and rat anti-MKI67 antibody (1:300; DAKO: M7249) and goat anti-rat, Alexa Fluor 568 (1:300, ThermoFisher Scientific: A-11077). One hundred cells were counted per section (n = 3) and the number of MKI67+ cells was analyzed. The results are shown as a percentage of MKI67+ cells per 100 cells. The statistical analysis was performed using a 2-tailed student t-test.

Centrosome analysis in vivo

Colonic sections of normal-appearing mucosa and tumors of Klf4fl/fl and Klf4ΔIS mice after AOM/DSS treatment were stained with antibodies against α-tubulin and γ-tubulin as described previously in this article. One hundred mitotic cells were counted in 3 animals per group, and the number of centrosomes was analyzed. The results are shown as a percentage of mitotic cells with 2 or >2 centrosomes. The statistical analysis was performed using a 2-tailed student t-test.

Cell lines and cell culture

MEFs Klf4+/+ and Klf4−/− were obtained as previously described (16) and maintained in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% FBS, 1% penicillin/streptomycin at 37°C with 5% CO2. All cell lines underwent routine morphology checks and were only passaged for 3 months. In addition, all cell lines were tested for Mycoplasma contamination. For overexpression experiments, Klf4−/− MEFs were transiently transfected with Klf4-HA and HA-control plasmids as described previously (17) using Lipofectamine 2000 reagent (ThermoFisher Scientific: 11668–019) according to the manufacturer’s instructions.

Immunofluorescence staining in vitro

Klf4+/+ and Klf4−/− cells were grown on coverslips, washed briefly with PBS, fixed with 3.7% formaldehyde for 30 minutes, washed 3 times with PBS, and blocked with 10% NDS in PBS for 1 hour at room temperature. Primary antibodies goat anti-KLF4 (1:100; R&D: AF3158), goat-anti-γ-tubulin (1:100, Santa Cruz Biotechnology; sc-7396), rabbit anti-p53 (1:100, Santa Cruz Biotechnology: sc-6243), and mouse anti-HA (1:1000, Covance: MMS-101P-500) were added to the blocking buffer and incubated at 4°C overnight. Twenty-four hours later, appropriate Alexa Fluor–labeled secondary antibodies (Molecular Probes) were added at a 1:300 dilution in blocking buffer for 30 minutes at 37°C, counterstained with Hoechst 33258 (ThermoFisher Scientific: H3569), mounted with Fluoromount Aqueous Mounting Medium (Sigma-Aldrich: F4680), and coverslipped. Slides were analyzed using a Nikon eclipse 90i microscope (Nikon Instruments Inc.) equipped with DS-Qi1Mc and DS-Fi1, CCD cameras (Nikon Instruments Inc.). The numbers of costained γ-tubulin and KLF4, γ-tubulin and p53, and γ-tubulin, KLF4, and p53 were determined in 100 cells per experiment (n = 3 or n = 5).

Biostatistical analysis

The analysis of in vitro and in vivo experiments was performed with a Student t-test or a Mann-Whitney nonparametric test. A value of p < 0.05 was considered significant. This analysis was performed using GraphPad Prism version 5.00 for Windows (GraphPad Software, San Diego, CA).

Results

KLF4 deficiency leads to accelerated tumorigenesis in CAC

Previously, we showed that KLF4 regulates the response of the intestinal epithelium to inflammatory insult in a DSS murine model (15) and plays a preventive role in sporadic colorectal carcinogenesis in a murine model utilizing azoxymethane (AOM) (14). Given the higher incidence of colorectal cancer in IBD patients (1, 21–23), we examined the role of KLF4 in colitis-associated colorectal cancer (CAC) using the well-established AOM/DSS model (24–26). Control mice (Klf4fl/fl) and mice with intestinal epithelium-specific deletion of Klf4 (Klf4ΔIS) were injected with AOM and then subjected to 2 rounds of DSS treatment as described in the Material and Methods section (Fig. 1A). At the end of the recovery phase after the second round of DSS treatment, we collected colonic tissues from both groups of mice for analysis. Initial gross histopathological assessment of hematoxylin and eosin (H&E) stained slides by our anatomic pathologists affirmed that colonic tissues of AOM/DSS-treated Klf4fl/fl mice exhibited adenomatous changes with signs of acute-to-chronic inflammation and rare presence of carcinomas in situ with microabscesses, while those of treated Klf4ΔIS mice showed numerous multifocal adenomatous changes with acute-to-chronic inflammation and frequent carcinomas in situ with microabscesses. Quantitative analysis showed that Klf4ΔIS mice developed significantly higher numbers of adenomatous polyps in the colon in comparison to control mice (Fig. 1B) (15.44 ± 2.03 and 5.81 ± 1.69 per mouse, respectively). In contrast there was no measurable difference in the size of the polyps between the groups (data not shown). As the shortening of the large intestine is one of the markers of chronic signs of inflammation we analyzed the colons of the treated mice (27, 28). The average colon length of treated Klf4ΔIS mice was shorter than that of control mice, indicating increased inflammation severity in the absence of KLF4 (Fig. 1C) (6.67 ± 0.14 and 7.67 ± 0.14 cm, respectively). Importantly Klf4ΔIS mice had a higher incidence of carcinomas in situ formation in comparison to the control group at end points (Fig. 1D). The examples of adenomatous polyps and carcinomas in situ for both groups of mice are presented in Figure 1E. Of note is that the progression from normal-appearing mucosa to adenomatous polyp and to formation of carcinomas in situ coincided with the loss of KLF4 expression in control mice (Fig. 2A, left panel). This suggests that the deletion of Klf4 from the intestinal epithelium (Fig. 2A, right panel) in conjunction with increased inflammatory changes may predispose Klf4ΔIS mice to earlier development of adenomas and carcinomas from AOM/DSS treatment in comparison to control mice.

Figure 1. Intestinal epithelium-specific deletion of Klf4 increases adenoma and carcinomas in situ formation in a murine model of inflammation-induced carcinogenesis.

Figure 1.

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(A) Schematics of the experimental design. (B) Quantification of adenomatous polyps in colonic tissues from Klf4fl/fl and Klf4ΔIS mice after AOM/DSS treatment (Klf4fl/fl, n = 16; and Klf4ΔIS, n = 18 mice). Data are represented as mean ± s.e.m, **p < 0.01 (2-tailed t-test). (C) Mean colon length of Klf4fl/fl and Klf4ΔIS mice after AOM/DSS treatment (Klf4fl/fl, n = 16; and Klf4ΔIS, n = 18 mice). Data are represented as mean ± s.e.m, ***p < 0.001 (2-tailed t-test). (D) Quantification of carcinomas in situ in colonic tissues from Klf4fl/fl and Klf4ΔIS mice after AOM/DSS treatment. (E) Representative H&E images of adenomatous polyps and carcinomas in situ from colonic tissues of Klf4fl/fl and Klf4ΔIS mice after AOM/DSS treatment. Scale bar, 100 μm.

Figure 2. Altered signaling pathways upon AOM/DSS treatment of Klf4fl/fl and Klf4ΔIS mice.

Figure 2.

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IHC staining for (A) KLF4, (B) phospho-β-catenin serine 552, (C) cyclin D1, (D) p65, and (E) phospho-ERK1/2 in normal-appearing mucosa, adenomatous polyps, and carcinomas in situ in colonic tissues of Klf4fl/fl (left panel) and Klf4ΔIS (right panel) mice after AOM/DSS treatment. Scale bar, 50 μm.

Loss of KLF4 activates the ERK pathway and increases proliferation

Previous studies established that WNT signaling plays a crucial role in the insurgence and progression of CAC (29–31). It has also been shown that KLF4 negatively regulates the WNT signaling pathway (32, 33). To investigate the role of KLF4 in WNT signaling in CAC we examined the patterns of expression of active, phosphorylated β-catenin at residue serine 552 and its transcriptional target cyclin D1 by immunohistochemistry (IHC) in the normal-appearing colonic mucosa, adenomatous polyps, and carcinomas in situ of age-matched Klf4fl/fl and Klf4ΔIS mice after AOM/DSS treatment. The results showed that there was a loss of membranous β-catenin and a progressive increase in the expression levels of nuclear phosphorylated β-catenin Ser552, accompanied by nuclear accumulation of cyclin D1, starting from the normal-appearing mucosa, adenomatous polyps, to carcinomas in situ in AOM/DSS mice, regardless of the KLF4 status (Figs. 2B and 2C). This suggests that progression from normal-appearing mucosa to carcinomas in situ from AOM/DSS treatment depends in part on these two proteins, and that the tumorigenic process is accelerated in mice lacking Klf4 in the intestinal epithelium given the higher tumor burden when compared to control mice. It has become clear that the pathways involved in the regulation of cytokines and growth factors play an important role during chronic inflammation and may enhance tumorigenesis (34). As such, the NF-κB pathway that regulates apoptosis, cell cycle progression, and differentiation has been shown to play a pivotal role in CAC development (35–38). Furthermore, increased nuclear β-catenin has been shown to positively regulate NF-κB activity during carcinogenesis (39, 40). Our previous studies showed that KLF4 is necessary for activation of the NF-κB pathway during DSS-induced colitis, and Klf4ΔIS mice had reduced levels of nuclear p65 relative to control mice (15). Here we examined the status of the NF-κB pathway in the AOM/DSS model. The results showed minimal p65 staining during development of adenomatous polyps and progression toward carcinomas in situ irrespective of the KLF4 status (Fig. 2D). Cytokine increase and β-catenin activation in CAC stimulate ERK pathway activity (41, 42). Previously, we showed that mice with intestinal epithelium deletion of Klf4 upon AOM treatment had increased levels of phospho-ERK1/2 in comparison to control mice (14). To further investigate the effect of intestinal epithelium-specific Klf4 deletion on the CAC formation and progression, we assessed phospho-ERK1/2 levels in colonic tissues from Klf4fl/fl and Klf4ΔIS mice after AOM/DSS treatment. As shown in the left panel of Figure 2E, the expression level of nuclear phospho-ERK1/2 in Klf4fl/fl was increased during the progression from adenomatous polyps to carcinomas in situ. There was a similar trend in Klf4ΔIS mice although it was more pronounced than in control mice (Fig. 2E, right panel). Sixty-seven percent of Klf4fl/fl mice had positive nuclear phospho-ERK staining, while at the same time, 100% of Klf4ΔIS mice had positive staining. The activation of WNT and ERK pathways resulted in increased proliferation of adenomatous polyps and carcinomas in situ in comparison to normal-appearing mucosa in both groups of mice, as shown by MKI67 (a marker for cell proliferation) staining (Fig. 3), although the number of MKI67-positive cells was higher within adenomatous polyps and carcinomas in situ in Klf4ΔIS mice as compared to Klf4fl/fl mice (66.33 ± 5.55 and 28.67 ± 2.19, and 72.33 ± 1.45 and 45.33 ± 3.84, respectively, and Figs. 3D and 3E). Taken together, these results indicate that loss of KLF4 in control mice treated with AOM/DSS is associated with temporal progression from normal-appearing mucosa to carcinomas in situ and induction of key signaling pathways. Mice with intestinal epithelium-specific deletion of Klf4 shared similar pathway activation during tumor progression with control mice but had a higher propensity for tumorigenesis in response to AOM/DSS treatment. These results suggest that KLF4 plays an important role in attenuating a number of key signaling pathways that lead to CAC.

Figure 3. The status of proliferation in colonic tissues of Klf4fl/fl and Klf4ΔIS mice after AOM/DSS treatment.

Figure 3.

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Representative IF images of (A) normal-appearing mucosa, (B) adenomatous polyps, and (C) carcinomas in situ in colonic tissues of Klf4fl/fl and Klf4ΔIS mice after AOM/DSS treatment. Hoechst labeled nuclei and MKI67+ marked proliferating cells. Scale bar, 50 μm. (D) Quantification of MKI67+ cells in adenomatous polyps and (E) in carcinoma in situ in colonic tissues of Klf4fl/fl and Klf4ΔIS mice after AOM/DSS treatment. One hundred cells were counted per section, n = 3. Data are represented as mean ± s.e.m, *p < 0.05 (t-test).

Loss of Klf4 causes centrosome amplification in CAC

A common feature of CAC is increased levels of chromosome instability caused by defects in chromosome cohesion, assembly of the microtubule spindle, and centrosome duplication (4, 43–45). Our previous studies showed that mouse embryonic fibroblasts (MEFs) null for Klf4 are genetically unstable, as evidenced by increased rates of cell proliferation, presence of DNA double strand breaks (DSBs), centrosome amplification, chromosome aberrations and aneuploidy (16, 17). Using centrosome amplification as a means to assess genetic instability, we performed immunofluorescence (IF) staining of α-tubulin (to label microtubules) and γ-tubulin (to label centrosomes) in colonic tissues of Klf4fl/fl and Klf4ΔIS mice treated with AOM/DSS. An example of such staining in normal-appearing mucosa and tumor sections of Klf4ΔIS mice treated with AOM/DSS is shown in Figure 4A. Quantification of the percentage of mitotic cells with 2 or more than 2 centrosomes (the latter as evidence of centrosome amplification) in normal-appearing colonic mucosa of Klf4fl/fl and Klf4ΔIS mice treated with AOM/DSS showed no difference between the two groups (Fig. 4B, left panel). In contrast, there was a significant increase in the percentage of mitotic cells with more than 2 centrosomes in tumor sections of Klf4ΔIS mice relative to Klf4fl/fl mice treated with AOM/DSS (Fig. 4B, right panel). These results suggest that deletion of Klf4 from the intestinal epithelium after AOM/DSS treatment led to centrosome amplification and chromosomal instability upon tumor formation.

Figure 4. Loss of KLF4 leads to centrosome amplification in adenomas after AOM/DSS treatment.

Figure 4.

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(A) Representative IF images of mitotic cells with 2 or >2 centrosomes in the normal-appearing mucosa (left panel) and tumor tissues (right panel) of Klf4ΔIS mice after AOM/DSS treatment. Hoechst labeled nucleus; α-tubulin-microtubule; and γ-tubulin-centrosomes. White arrows indicate centrosomes. Scale bar, 10 μm. (B) Quantification of mitotic cells with 2 or >2 centrosomes in the normal-appearing mucosa (left panel) and tumor tissues (right panel) of Klf4fl/fl and Klf4ΔIS mice after AOM/DSS treatment. One hundred cells were counted per mouse, n = 3. Data are represented as mean ± s.e.m, *p < 0.05 (t-test).

KLF4 regulates localization of p53 to the centrosomes

The above results showed that deficiency in KLF4 led to centrosome amplification, which may promote tumorigenesis. In mammalian cells, p53 controls proper timing of centrosome separation and defects in p53 promote genetic instability through a mechanism that involves formation of cells with supernumerary centrosomes (46, 47). Additionally, p53 is associated with centrosomes during mitosis and regulates their segregation by a mechanism that is yet to be identified (48). One of the hallmarks of CAC is dysregulation of p53 that directly correlates to aneuploidy. Furthermore, our preliminary results obtained from Isobaric tag for relative and absolute quantitation (iTRAQ) analysis (data not shown) of centrosome components showed significant reduction of p53 levels in Klf4−/− centrosomal fraction in comparison to the same fraction from Klf4+/+ MEFs. Therefore, we examined whether KLF4 regulates p53 localization to the centrosomes. Using IF staining, we demonstrated that in MEFs with wild type Klf4 (Klf4+/+), KLF4 was localized to the centrosomes (Fig. 5A, top panels) and was absent in Klf4−/− MEFs (Fig. 5A, bottom panels). We then co-stained Klf4+/+ and Klf4−/− MEFs for p53 and γ-tubulin. As shown in the top panels of Fig. 5B, p53 was localized to the centrosomes in Klf4+/+ MEFs. In contrast, p53 was no longer localized to the centrosomes in the absence of KLF4 and cells exhibited an increased number of centrosomes (Fig. 5B, bottom panels). Quantification showed that 76 ± 4% of Klf4+/+ MEFs cells displayed centrosome staining of KLF4, 92 ± 1.5% stained for p53, and 76 ± 4% stained for both KLF4 and p53 (Fig. 5C). Finally, overexpression of Klf4 in Klf4−/− MEFs resulted in re-localization of p53 to the centrosomes (Fig. 6A), and this effect was solely dependent on KLF4 localization to the centrosomes (Fig. 6B). Taken together, these observations suggest that KLF4 protects against the progression of CAC by assisting p53 to regulate centrosome duplication and therefore genetic stability.

Figure 5. KLF4 is necessary for localization of p53 to the centrosomes in mouse embryonic fibroblasts (MEFs).

Figure 5.

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(A) Representative IF images of KLF4 and γ-tubulin (centrosome) in Klf4+/+ and Klf4−/− MEFs. (B) Representative IF images of p53 and γ-tubulin in Klf4+/+ and Klf4−/− MEFs. Scale bar, 50 μm. (C) Quantification of percentage of centrosome costaining with KLF4 or p53 or KLF4 and p53 in Klf4+/+ MEFs. One hundred cells were counted per field, n = 5. Data are represented as mean ± s.e.m.

Figure 6. Overexpression of Klf4 reestablishes p53 localization to the centrosomes in MEFs.

Figure 6.

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(A) Representative IF images of p53 and γ-tubulin (centrosome) in Klf4−/− MEFs overexpressing Klf4. Centrosomes for which p53 localization was reinstated are indicated by white arrows. Scale bar, 50 μm. (B) Quantification of percentage of centrosome costaining with KLF4 (left panel), p53 (middle panel), and KLF4 and p53 (right panel) in Klf4−/− and Klf4−/− with Klf4 overexpression MEFs. One hundred cells were counted per field, n = 5. Data are represented as mean ± s.e.m, *p < 0.05 (Mann-Whitney test).

Discussion

Recent studies demonstrated that KLF4 guards against the development of colorectal cancer. In this study, we investigated the role of KLF4 in CAC development and progression. To our knowledge, this is the first study showing a key role for KLF4 in the development of CAC.

In this study, we showed that KLF4 inhibits tumorigenesis by maintaining genetic stability. First, Klf4ΔIS mice treated with AOM/DSS developed more adenomatous polyps and carcinomas in situ in comparison to treated control mice (Fig. 1). Second, Klf4ΔIS mice had increased inflammation in colonic tissues as compared to control mice. Third, the progression from normal-appearing mucosa to adenomatous polyps to carcinomas in situ was accompanied by loss of KLF4 expression in control mice and the progression was exacerbated in mice with intestinal epithelium-specific deletion of Klf4 (Fig. 2). The latter was substantiated by the increased proliferation as shown by MKI67 stain in adenomatous polyps and carcinomas in situ from AOM/DSS-treated Klf4ΔIS mice relative to treated control mice and nuclear phospho-ERK in tumors derived from AOM/DSS-treated Klf4ΔIS mice relative to treated control mice (Figs. 2 and 3). Fourth, mice with Klf4ΔIS displayed more mitotic cells with supernumerary centrosomes in the adenomatous polyps relative to that in control mice (Fig. 4). We also showed that both KLF4 and p53 localize to the centrosomes of wild type MEFs and that p53 does not localize to the centrosomes in Klf4−/− MEFs (Fig. 5). Finally overexpression of Klf4 in Klf4−/− MEFs restored p53’s centrosomal localization (Fig. 6). Taken together, our data suggest that KLF4 plays an important role in regulating CAC by cooperating with p53 to maintain genetic stability.

Our observation that KLF4 expression is decreased during the tumorigenic process fits well with previous publications (11–13, 20). Our data showed that both AOM/DSS-treated Klf4fl/fl and Klf4ΔIS mice had inflammation and developed adenomatous polyps and carcinomas in situ with Klf4ΔIS mice having a higher tumor burden. This suggests that a lack of Klf4 may predispose mice to early CAC formation relative to control mice. Similar to our observations, Li and colleagues showed that inactivation of Klf4 within the population of gastric progenitors cells increases the rate of tumors formation in comparison to control mice (49). Furthermore, they demonstrated a negative correlation between KLF4 and FoxM1 levels in mouse and human gastric cancer, thereby strengthening the concept of KLF4 being a tumor suppressor. On the other hand, it has been shown that the expression levels of KLF4 are increased upon injury to pancreatic acinar cells in the context of KRAS mutation and that KLF4 positively regulates expression of ductal markers during pancreatic neoplasia formation (50). These results suggest that increased levels of KLF4 positively regulate the development of pancreatic cancer. Therefore, the distinct roles of KLF4 as a tumor suppressor or oncoprotein depend on its transcriptional targets and/or protein interaction network within the type of the tissues that the cancer originates.

Reports have shown that increased and persistent inflammation during CAC supports the initiation of tumorigenesis by oxidative stress-induced mutations and promotes genetic instability (6, 7, 51). Importantly, we previously demonstrated that KLF4 regulates genetic stability in MEFs. Klf4−/− MEF cells exhibited an increased number of double-strand breaks in DNA and γ-H2AX levels, abnormal centrosome numbers, aneuploidy, and micronuclei in comparison to Klf4+/+ MEFs (17, 18). Our recent publication, using 2 independent models of colorectal carcinogenesis, similarly demonstrated that KLF4 plays a protective role in tumor development (14). Colonic tissues of Klf4ΔIS/ApcMin/+ mice showed increased chromosomal instability and aberrant mitosis. Furthermore, in these models we showed that KLF4 was crucial in regulating the DNA damage repair pathways (14). In the current CAC model, we demonstrated that Klf4ΔIS mice treated with AOM/DSS had a significantly increased number of mitotic cells with supernumerary centrosomes (Fig. 4). This and previous observations support the notion that KLF4 is an important regulator of genetic stability in vitro and in vivo. Interestingly, it has been shown that KLF14 maintains genetic stability by regulating Polo-like kinase 4 expression and centrosome integrity, and that KLF14-KO mice are more susceptible to AOM/DSS tumorigenesis (52).

Genetic stability depends on the accuracy of chromosome segregation by the mitotic spindle. Thus, aberrant expression or function of factors involved in centrosomes regulation can lead to their dysfunction or an abnormal number and ultimately to chromosomal instability, a hallmark of tumorigenesis (4). The role of p53, a tumor suppressor, has long been studied in the context of centrosome amplification and chromosome aneuploidy (53). It has been shown that the absence of p53 may be directly responsible for driving centrosome amplification by activation of the CDK inhibitor p21, which drives centrosome duplication in G1/S arrested cells (54). On the other hand, p53 may be directly required at the centrosome to mediate its duplication at the G1/S transition (55). Here we showed that Klf4ΔIS mice treated with AOM/DSS displayed supernumerary centrosomes within the tumor sections (Fig. 4) and that both KLF4 and p53 localized to the centrosomes (Fig. 5). Our previous studies showed that lack of KLF4 led to genetic instability and increased levels of p53 (17, 18). In the current study, we showed that despite increased protein levels of p53 in Klf4−/− MEFs, p53 did not localize to the centrosomes, and that KLF4 was necessary for its localization (Figs. 5 and 6). As p53 fails to localize to the centrosome in the absence of KLF4 (Fig. 5) and KLF4 is not observed at the centrosomes without p53, our data suggest that KLF4 is responsible for initiating the re-localization of p53 to the centrosome, but not in maintaining its to this region. At present, the molecular mechanism by which KLF4 regulates p53 localization to the centrosomes is not well understood. Our results provide a novel model of KLF4-p53 interaction and the role for KLF4 and p53 in the maintenance of genetic stability during development of CAC.

Implications: Taken together, these results indicate that KLF4 plays a protective role against progression of CAC by guarding against genetic instability.

Acknowledgments

We thank Dr. Amr Ghaleb, PhD, from the Department of Pathology at Stony Brook University for the assistance with the animal experiments and the Department of Pathology at Stony Brook University for technical assistance in histopathological analysis and Donglei Yin, PhD, from the Department of Applied Mathematics and Statistics at Stony Brook University for the assistance with statistical analysis. This work was supported by grants from the National Institutes of Health awarded to V.W.Y. (CA084197 and DK052230).

Footnotes

The authors declare no potential conflicts of interest.

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